A flow cytometric method for measuring neutralization of HIV-1 subtype B and E primary isolates

Abstract

Background: Clinical trials testing candidate human immunodeficiency virus type 1 (HIV-1) vaccines have required the use of HIV neutralization assays to detect responses to specific geographic subtypes of HIV-1. The variability in results seen with current p24 neutralization assay endpoints prompted us to assess the utility of flow cytometry for monitoring the neutralization of HIV-1 primary isolates. Methods: A modified neutralization assay was performed using CD8-depleted peripheral blood mononuclear cells (PBMC). The cells were fixed, permeabilized, stained with a directly conjugated HIV-1 p24 monoclonal antibody, and analyzed by flow cytometry. HIV-1 subtype B' and E primary isolates were tested using pooled sera or plasma from subtype B' or E infected patients. Results: Primary isolate cultures (without neutralizing antibody) showed from 18% to 42% p24+ cells, depending on the virus. Less than 0.2% p24+ cells were detected in uninfected cultures. Subtype-specific neutralization of viruses was observed using plasma or serum pools; neutralization ranged from 0% to 99% reduction of infected cells. Conclusions: Flow cytometric detection of intracellular HIV-1 p24 can be used as an endpoint assay to assess neutralization of HIV-1 subtypes B' and E primary isolates. This enumerative method has the advantage of identifying intracellular p24 in specific subsets at an early culture timepoint. It also provides an alternative quantitative end-point for HIV neutralization assays. (C) 2000 Wiley-Liss, Inc.